1.1 Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, certain embodiments concern methods and compositions comprising DNA segments, and proteins derived from bacterial species. More particularly, the invention provides cna and cna-derived nucleic acid compositions comprising a collagen (Col) binding protein (CBP) from Staphylococcus aureus and the corresponding peptide epitopes and protein sequences comprising native and synthetically-modified Col binding site domains. Various methods for making and using these DNA segments, DNA segments encoding synthetically-modified ligand binding site domains, and native and synthetic proteins are disclosed, such as, for example, the use of DNA segments as diagnostic probes and templates for protein production, and the use of proteins, fusion protein carriers and peptides in various pharmacological and immunological applications.
1.2 Description of the Related Art
1.2.1 Colonization by Staphylococcus aureus
S. aureus cells can colonize many different host tissues and cause various types of infections such as endocarditis, pneumonia, wound infections, osteomyelitis, and septic arthritis. Adherence of staphylococci to host tissues involves a family of adhesions that recognize extracellular matrix components and which have been named MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules) (Patti et al., 1994a).
The expression of specific MSCRAMMs appears to be needed for the colonization of different types of tissues. For example, staphylococcal strains recovered from the joints of patients diagnosed with septic arthritis or osteomyelitis almost invariably express a CBP, whereas significantly fewer isolates obtained from wound infections express this adhesin. (Switalski et al., 1993a) Similarly, S. aureus strains isolated form the bones of patients with osteomyelitis often have an MSCRAMM recognizing the bone-specific protein, bone sialoprotein (BSP) (Ryden et al., 1989).
The cloning, sequencing, and expression of a gene cna, encoding a S. aureus CBP has been previously reported (Patti et al., 1992). The cna gene encodes an 133-kDa adhesin that contains structural features characteristic of surface proteins isolated from Gram-positive bacteria. It has been demonstrated that the CBP is required and sufficient for the adherence of S. aureus to Col-coated artificial substrates as well as to cartilage, a tissue rich in type II Col (Switalski et al., 1993a). All strains expressing the CBP were able to adhere to cartilage, whereas those strains lacking the MSCRAMMs did not adhere. Preincubation of S. aureus with polyclonal antibodies raised against the purified adhesin or saturation of the cartilage substrata with soluble recombinant CBP resulted in a complete inhibition of bacterial attachment (Switalski et al., 1993a).
S. aureus colonization of the articular cartilage within the joint space appears to be an important factor contributing to the development of septic arthritis. The importance of the CBP in the pathogenesis of septic arthritis was examined by comparing the virulence of two sets of S. aureus isogenic mutants in an animal model (Patti et al., 1994b). Greater than 70% of mice injected with CNA.sup.+ strains (i.e. a clinical isolate expressing the CBP or a negative strain into which the cna gene had been introduced) developed clinical signs of arthritis, whereas less than 27% of the animals showed symptoms of disease when injected with CNA.sup.- strains (i.e. a strain lacking the cna gene or a strain in which the cna gene or a strain in which the cna gene had been inactivated through homologous recombination). Taken together these results demonstrate that the CBP plays an important role in the pathogenesis of septic arthritis induced by S. aureus.
Recently, the ligand-binding site has been localized within the N-terminal half of the CBP (Patti et al., 1993). By analyzing the Col binding activity of recombinant proteins corresponding to different segments of the MSCRAMM, a 168-amino-acid long protein fragment (corresponding to amino acid residues 151-318) that had appreciable Col binding activity was identified. Short truncations of this protein in the a N- or C terminus resulted in a loss of ligand binding activity but also resulted in conformational changes in the protein as indicated by circular dichroism spectroscopy. These results raised the possibility that the ligand-binding activity but also resulted in conformational changes in the protein as indicated by circular dichroism spectroscopy. These results raised the possibility the ligand-binding site of the MSCRAMM is contained within a short segment of amino acids and that flanking sequences are required for the proper folding of these residues in the ligand-binding site.
1.2.2 Role of S. aureus CBP in Human Disease
Hematogenously acquired bacterial arthritis remains a serious medical problem. This rapidly progressive and highly destructive joint disease is difficult to eradicate with less than 50% of the infected patients failing to recover without serious joint damage. S. aureus is the predominant pathogen isolated from adult patients with hematogenous (primary) and secondary osteomyelitis (Waldvogel and Vasey 1980), while also causing up to 90% of the cases of acute hematogenous osteomyelitis in otherwise healthy children (Cole 1982). Scanning electron microscopy studies have shown S. aureus to be intimately associated with cartilage and bone tissue retrieved from the site of infection. Additional microscopic evidence suggests the predominate attachment and subsequent colonization of cartilaginous rather than synovial surfaces (Voytek et al. 1988). An analysis of S. aureus strains isolated from patients diagnosed with osteomyelitis and septic arthritis revealed that almost all of the isolates contained a Col adhesin. In contrast, only one-third of the S. aureus strains isolated from patients with soft tissue infections expressed the Col adhesin (Switalski, Patti et al. 1993). These observations suggest that cell surface expression of the Col adhesin is an important virulence factor in staphylococcal mediated osteomyelitis and septic arthritis. Moreover, it has been observed that cartilage degradation following staphylococcal joint infection can be attributed to a direct interaction between bacteria and cartilage (Smith et al. 1982) in addition to the inflammatory response of the host (Smith et al. 1987). Individuals who require more than 6 days for the synovial fluid to become free of microorganisms typically result in poor clinical outcome (Ho and Su 1982). Poor outcomes include permanent disability with limited motion or persistent pain in the affected joint. Therefore, by inhibiting the initial attachment of bacteria to cartilage, the likelihood of subsequent joint destruction may be diminished.
1.3 Deficiencies in the Prior Art
It is clear that while several approaches to the treatment of bacterial diseases have experienced some success, many problems remain, including antibiotic resistance, variability of antigens between species and species variation through mutation of antigens, as well as the need to protect susceptible groups such as young children, the elderly and other immunocompromised patients. Thus, there exists an immediate need for an effective treatment for S. aureus infection, and vaccines against this pathogen.